The use of MetaboReport is pretty simple. You only need to follow the user guide in each tab to perform your data analysis.
MetaboReport accepts a variety of outputs produced by different software tools, such as XCMS, MSDIAL, and Compound Discoverer. The output table can be submitted to MetaboReport with very little modification.
Like any other software tools, MetaboReport requires at least two pieces of information for metabolomics data analysis, i.e., Peak area and sample metadata.
Peak Area: is the area under a peak (peak area count). It is a measure of the concentration of the peak it represents. Sometimes, peak intensity or peak height maybe used to represent the the concentration of the peak, depending on the software tools used for data processing.
Sample Metadata: or sample label or group label. It is used to describe the sample.
There are two ways to prepare input data for MetaboReport:
1. The peak area and metadata are in the same table. You only need to upload one single table for data analysis.
2. The peak area table and metadata table are two separated tables. You need to upload both of them for data analysis.
This manual will guide you prepare the input data table for MetaboReport.
Here an example is used to show you how to prepare the input data table for MetaboReport.
Suppose you want to compare the metabolic profiles between a group of
wild type (WT) and Mutant (MU) mice;
WT and MU are the metadata, meaning each
sample is labeled with either a WT or
Mu metadata.
Meanwhile, you believe that gender may also play an important role in
your study, so you introduced another set of metadata in your study,
namely Male and Female.
In order to monitor, evaluate and correct system variations in the
studies, you also introduced a set of pooled QC (QC)
samples.
This study contains two groups of metadata, i.e., WT, MU and QC in group 1, and Male, Female and QC in group 2. As such each sample is labeled with two metadata. For instance, Sample3 is WT and Male, and Sample13 is QC and QC (Table 1).
Table 1. Experiment design of a metabolomics study.
| Sample | Sample Label 1 | Sample Label 2 |
|---|---|---|
| Sample1 | WT | Male |
| Sample2 | WT | Female |
| Sample3 | WT | Male |
| Sample4 | WT | Female |
| sample5 | WT | Male |
| Sample6 | WT | Male |
| Sample7 | MU | Female |
| Sample8 | MU | Male |
| Sample9 | MU | Female |
| sample10 | MU | Male |
| sample11 | MU | Female |
| sample12 | MU | Female |
| Sample13 | QC | QC |
| sample14 | QC | QC |
| sample15 | QC | QC |
| sample16 | QC | QC |
There are in total 16 samples in this study (Table 1). After LC-MS analysis and data processing, you will a data table as shown in Table 2. This is a typical sample-feature table, in which each row represents one mass feature, and columns are different descriptors of the feature.
Table 2. An example of sample-feature table.
| mz | Retention Time | Identification | PeakArea of Sample1 | PeakArea of Sample2 | …. | PeakArea of Sample16 |
|---|---|---|---|---|---|---|
| 133.014 | 3.23 | Malic acid | 123000 | 120000 | … | 801000 |
| 144.012 | 2.44 | Unknown | 678555 | 666000 | … | 1200 |
| 333.331 | 10.18 | Unknown | 3400 | 2000 | … | 560000 |
| 234.446 | 5.52 | Unknown | 45000 | 55000 | … | 46000 |
| … | … | … | … | … | … | … |
| 1034.552 | 25.33 | Tomatine | 4780 | 4300 | … | 12000 |
Table 2 will be modified in order to to prepare the required input data table for MetaboReport.
As discuss above, there are two ways to prepare the input data table for MetaboReport.
The method one is simple, you only need to modify all the peak area columns shown in Table 2.
For instance, the PeakArea of Sample1 needs to be
modified to Sample_WT_Male_1. See Table 3 for details.
Explanation:
- Keyword
Sampleneed to be added in Peak Areas columns are so that MetaboReport will know which columns contain peak area information. Pay attention that the keywordSampleis case sensitive.- Sample1 has two metadata (see Table 1), so each
metadata, i.e. WT and MALE, are added afterSamplekeyword, and they are separated by_. Note thatMetaDataare also case sensitive, WT and wt are different.- A
unique numberis added after metadata, which make each sample different from its replicates.- all the other columns are not important or necessary for MetaboReport. You don’t have to delete them, you can leave them as they are.
Table 3. Sample-Feature table for MetaboReport prepared by method 1.
| mz | Retention Time | Sample_WT_Male_1 | Sample_WT_Female_2 | …. | Sample_QC_QC_16 |
|---|---|---|---|---|---|
| 133.014 | 3.23 | 123000 | 120000 | … | 801000 |
| 144.012 | 2.44 | 678555 | 666000 | … | 1200 |
| 333.331 | 10.18 | 3400 | 2000 | … | 560000 |
| 234.446 | 5.52 | 45000 | 55000 | … | 46000 |
| … | … | … | … | … | … |
| 1034.552 | 25.33 | 4780 | 4300 | … | 12000 |
Note that you can also name your raw data in this way before you perform data analysis, then the generated sample-feature table can be directly used by Metaboreport.
The second method requires to prepare an additional metadata table.
For peak area table:
- Keyword
Sampleneed to be added in Peak Areas columns are so that MetaboReport will know which columns contain peak area information. Pay attention that the keywordSampleis case sensitive.- A
unique numberis needed after theSampleKeyword. This is make each sample unique.- all the other columns are not important or necessary for MetaboReport. You don’t have to delete them, you can leave them as they are.
- The peak area table can be also prepared exactly in the same way as shown in Mehtod 1.
See Table 4 for details.
Table 4. Peak area table for MetaboReport prepared by method 2.
| mz | Retention Time | Identification | Sample1 | Sample2 | …. | Sample16 |
|---|---|---|---|---|---|---|
| 133.014 | 3.23 | Malic acid | 123000 | 120000 | … | 801000 |
| 144.012 | 2.44 | Unknown | 678555 | 666000 | … | 1200 |
| 333.331 | 10.18 | Unknown | 3400 | 2000 | … | 560000 |
| 234.446 | 5.52 | Unknown | 45000 | 55000 | … | 46000 |
| … | … | … | … | … | … | … |
| 1034.552 | 25.33 | Tomatine | 4780 | 4300 | … | 12000 |
For Metadata table:
You need to prepare a seperate table to descripe the metadata of each sample. See Table 5. The header names are not important. You can give any names, but the first column must be sample. Note that
MetaDataare case sensitive, so pay attention to the consistancy.
Table 5. Metadata table for MetaboReport prepared by method 2.
| Sample | Sample Label 1 | Sample Label 2 |
|---|---|---|
| Sample1 | WT | Male |
| Sample2 | WT | Female |
| Sample3 | WT | Male |
| Sample4 | WT | Female |
| sample5 | WT | Male |
| Sample6 | WT | Male |
| Sample7 | MU | Female |
| Sample8 | MU | Male |
| Sample9 | MU | Female |
| sample10 | MU | Male |
| sample11 | MU | Female |
| sample12 | MU | Female |
| Sample13 | QC | QC |
| sample14 | QC | QC |
| sample15 | QC | QC |
| sample16 | QC | QC |
Notice that: the sample names in Table 4 and Table 5 don’t have to be the same, but the samples in peak area table and metadata table must be in the same order; otherwise, wrong metadata will be assigned to the samples. See Figure 2 below.
Figure 1. Pay attention that the samples in peak area table and metadata table must be in the same order.
The most common mistakes you may encounter during data analysis using MetaboReport is the data format issue.
Note that
Metadata is canse sensitive, therefore WT, Wt and Wt, and wt will be treated as different metadata, so be consistant with the metadata.
Sample is
reserved keyword, which is used to help MetaboReport software to recognize peak area columns.QC is
reserved keyword, which is used to represent QC samples only.Pay attention to white spaces.
WTandWTwith one space will be treated as different metadata.